The invention is in the field of conductive polymers, particularly conductive nucleic acids, such as DNA, as well as methods for producing and using such compounds.
Polymeric molecular conductors are known. For example, some naturally occurring proteins facilitate electron transfer in such fundamental biological processes as photosynthesis and respiration. Electron transfer in such systems is generally understood to occur as the result of quantum mechanical xe2x80x98tunnellingxe2x80x99 of electrons along pathways, molecular orbitals, that connect one atom to the next in the polymer.
It has been proposed that the stacked aromatic bases of DNA may act as a xe2x80x98xcfx80-wayxe2x80x99 for the transfer of electrons (Dandliker et al., 1997; Hall et al., 1996; Arkin et al., 1996). This proposal is based on a theory that the stacked arrangement of bases on complementary strands juxtaposes the shared electrons in the xcfx80 orbitals of the aromatic nitrogen bases, facilitating quantum mechanical tunnelling along the stack of base pairs. A number of experiments have supported the view that this effect exists, while other experiments have provided contrary evidence that the effect is limited or non-existent.
For example, experiments have been reported to demonstrate that photoinduced electron transfer may occur between two metallointercalators tethered at either end of a 15-base pair DNA duplex (Murphy et al., 1993). On the other hand, kinetic analysis of distance-dependent electron transfer in a DNA hairpin has been used to show that DNA is a poor conductor, only somewhat more effective than proteins as a conductor of electrons (Lewis et al., 1997; Taubes, 1997).
U.S. Pat. Nos. 5,591,578; 5,705,348; 5,770,369; 5,780,234 and 5,824,473 issued to Meade et al. on, respectively, Jan. 7, 1997, Jan. 6, 1998, Jun. 23, 1998, Jul. 14, 1998 and Oct. 20, 1998 (and incorporated herein by reference) disclose nucleic acids that are covalently modified with electron transfer moieties along the nucleic acid backbone. Meade et al. teach that such modifications are necessary for nucleic acids to efficiently mediate electron transfer.
The theory of xcfx80-orbital-mediated conductance along a nucleic acid duplex suggests that, as a precondition, such conductance requires a stable duplex with stacked base pairs. The effect on duplex stability of the binding of metal ions to nucleic acids, particularly DNA, has been studied extensively for nearly 40 years. In general, cations that bind primarily to the phosphate backbone will stabilize the duplex conformation, whereas those that bind to the bases will tend to denature the duplex. These effects are readily demonstrated with thermal denaturation profiles (Tm measurements). Experiments of this sort show that most monovalent cations, such as Na+, which tend to interact with the phosphate backbone, stabilize the duplex. This effect is reflected in the finding that there is approximately a 12xc2x0 C. increase in Tm for each 10-fold increase in monovalent cation concentration (Marmur and Doty 1962). An exception to this general principle is Ag+, which binds tightly to nitrogen bases, destabilizes the duplex, and therefore decreases the duplex Tm (Guay and Beauchamp 1979). Similarly, multivalent ions, particularly polyamines, which interact with the phosphate backbone are very effective duplex stabilizers.
For divalent metal cations, a series can be written in increasing order of DNA destabilization: Mg2+, Co2+, Ni2+, Mn2+, Zn2+, Cd2+, Cu2+ (Eichorn 1962; Eichorn and Shin 1968). At one end of the spectrum, Mg2+ increases the Tm at all concentrations; at the other end of the spectrum, sufficiently high concentrations of Cu2+ will lead to complete denaturation of the duplex at room temperature (Eichorn and Shin 1968). This series also correlates with the ability of the divalent cations to bind to the bases (Hodgson 1977; Swaminathan and Sundaralinghamn 1979).
Cations are also involved in promoting several other structural transitions and dismutations in nucleic acids. It has previously been reported that Zn2+ and some other divalent metal ions bind to duplex DNA at pHs above 8 and cause a conformational change (Lee et al., 1993). Preliminary characterization of the resulting structure incorporating zinc showed that it retained two antiparallel strands but that it was distinct from normal xe2x80x98Bxe2x80x99 DNA: it did not bind ethidium bromide, it appeared to lose the imino protons of both A-T and G-C base pairs upon addition of a stoichiometric amount of Zn2+, and it contained at least 5% fewer base pairs per turn than xe2x80x98Bxe2x80x99 DNA.
The invention provides an electrical conductor comprising an electron source electrically coupled to a conductive metal-containing nucleic acid duplex (CM-CNA). An electron sink may also be electrically coupled to the CM-CNA. The CM-CNA comprises a first strand of nucleic acid and a second strand of nucleic acid. The first and the second nucleic acid strands include a plurality of nitrogen-containing aromatic bases covalently linked by a backbone (the backbone may be made up of phosphodiester bonds, as in DNA or RNA, or alternative structures as discussed below). The nitrogen-containing aromatic bases of the first nucleic acid strand are joined by hydrogen bonding to the nitrogen-containing aromatic bases of the second nucleic acid strand. The nitrogen-containing bases on the first and the second nucleic acid strands form hydrogen-bonded base pairs in stacked arrangement along the length of the CM-CNA. At least some, and preferably each, of the hydrogen-bonded base pairs comprises an interchelated divalent metal cation coordinated to a nitrogen atom in one of the aromatic nitrogen-containing bases.
The electron source electrically coupled to the CM-CNA may be an electron donor molecule capable of donating an electron to the conductive metal-containing nucleic acid duplex. Similarly, the electron sink may be an electron acceptor molecule capable of accepting an electron from the CM-CNA. The electron donor molecule may be a fluorescent molecule, such as fluorescein. Similarly, the electron acceptor molecule may be a fluorescent molecule, such as rhodamine. It will be appreciated that some molecules may act both as electron donors and electron acceptors in various embodiments of the invention.
The CM-CNA may be made of deoxyribonucleic acid strands, which together produce metal-containing DNA (xe2x80x9cM-DNAxe2x80x9d). The nitrogen-containing aromatic bases in the nucleic acid may be the naturally occurring bases: adenine, thymine, guanine and cytosine.
In various embodiments, the divalent metal cation used to make CM-CNA may be Zn2+, Co2+, or Ni2+. Some divalent metal cations will not produce CM-CNA, and the present invention provides simple assays to determine whether a particular divalent metal cation will work to produce CM-CNA.
The divalent metal cations may be substituted for the imine protons of aromatic nitrogen-containing bases in the CM-CNA. In one embodiment, the divalent metal cations may be substituted for the N3 imine proton of thymine, or the imine protons of the N1 nitrogen atom of guanine.
The invention provides a method for making conductive metal-containing nucleic acid duplexes. A nucleic acid duplex is subjected to basic conditions in the presence of a divalent metal cation under conditions effective to form a conductive metal-containing nucleic acid duplex. Electron sources and sinks may be electrically coupled to the conductive metal-containing nucleic acid duplex, which may take the form of various embodiments discussed above.
The invention provides a method for detecting the formation of conductive metal-containing nucleic acid duplexes from first and second nucleic acid strands. The nucleic acid strands are mixed under conditions which allow complementary stands to hybridize and subjected to basic conditions in the presence of a divalent metal cation under conditions effective to form a conductive metal-containing nucleic acid duplex if the first and second strands are complementary. An electron source is provided electrically coupled to the conductive metal-containing nucleic acid duplex. Conductance of electrons between the electron source and the conductive metal-containing nucleic acid duplex is then tested to determine whether a CM-CNA has formed. The CM-CNA may take the form of various embodiments discussed above.
CM-CNAs of the invention may be used to carry electrons. They may also be used to raise antibodies in an animal, producing antibodies to CM-CNA. This latter use takes advantage of the finding that in some embodiments and under certain conditions CM-CNAs may be nuclease resistant.